Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are branch-point intermediates in the isoprenoid biosynthetic pathway. These isoprenoids are synthesized via a series of sequential condensations of five-carbon units catalyzed by the enzymes FPP synthase and GGPP synthase, respectively. FPP sits at the branch-point between sterol and longer chain non-sterol synthesis. GGPP is a precursor for ubiquinone synthesis and in plants, serves as the precursor for carotenoids, diterpenes, and chlorophylls. FPP and GGPP also serve as isoprene donors in the isoprenylation of proteins catalyzed by the enzymes farnesyl protein transferase (FPTase) and geranylgeranyl protein transferase (GGPTase) I and II (See Reiss, Y., et al., Cell. 1990, 62, 81-88; Moomaw, J. F. and Casey, P. J., J. Biol. Chem, 1992, 267, 17438-17443; Yokoyama, K. and Gelb, M. H., J. Biol. Chem, 1993, 268, 4055-4060; and Armstrong, S. A., et al., J. Biol. Chem., 1993, 268, 2221-12229). Isoprenylation of proteins, in particular small GTPases, serves to ensure proper intracellular localization and function.
While expression of FPP synthase has been shown to be regulated by sterol availability (Spear, D. H., et al., J. Biol. Chem, 1992, 267, 14462-14469), GGPP synthase appears to be regulated in a sterol-independent manner. The gene encoding human GGPP synthase has been cloned and GGPP synthase mRNA is expressed ubiquitously, with highest levels found in the testis (Ericsson, J., et al., J. Lipid Res, 1998, 39, 1731-1739). In rat thyroid cells, GGPP synthase expression is upregulated, coincident with cellular proliferation, following the stimulation of cells with thyrotropin and insulin (Fuse, M., et al., Biochem. Biophys. Res. Commun., 2004, 315, 1147-1153). In addition, GGPP synthase was first cloned in mice as a result of its identification as one of the genes upregulated in ob/ob mice, a model of obesity and insulin resistance (Vicent, D., et al., Mol. Cellular Biol, 2000, 20, 2158-2166). Thus alterations in levels of GGPP appears important in both physiological and pathophysiological processes and a method to experimentally manipulate intracellular GGPP levels would provide further understanding of these processes.
Nitrogen-containing bisphosphonates, including alendronate, pamidronate, and zoledronic acid, have been shown to inhibit FPP synthase (van Beek, E., et al., Biochem. Biophys. Res. Commun., 1999, 264, 108-111; Keller, R. K. and Fliesler, Biochem. Biophys. Res. Commun., 1999, 266, 560-563; and Bergstrom, J. D., Bostedor, R. G., et al., Arch. Biochem. Biophys., 2000, 373, 231-241). This class of drugs is used to inhibit bone resorption in a number of diseases, including osteoporosis, tumor-associated bone disease, and Paget's disease. The aminobisphosphonates, by depleting cells of both FPP and GGPP, prevent the farnesylation and geranylgeranylation of small GTPases (Luckman, S. P., Hughes, D. E., et al., J. Bone Miner. Res., 1998, 13, 581-589; Reszka, A. A., Halasy-Nagy, et al., J. Biol. Chem., 1999, 274, 34967-34973; and Benford, H. L., Frith, J. C., et al., Mol. Pharmacol., 1999, 56, 131-140). It appears that the depletion of GGPP, with subsequent prevention of geranylgeranylation is the critical mechanism underlying the effects of the aminobisphosphonates. In specific, it has been suggested that the loss of activity of geranylgeranylated proteins, such as cdc42, Rac, and Rho in osteoclasts, is directly related to the antiresorptive effects as restoration of geranylgeranylation blocks the effects of the aminobisphosphonates on osteoclasts (Reszka, A. A., Halasy-Nagy, et al., J. Biol. Chem., 1999, 274, 34967-34973; Fisher, J. E., Rogers, M. J., et al., Proc. Nat. Acad. Sci. U.S.A., 1999, 96, 133-138; and van beek, E., Lowik, et al., J. Bone Miner. Res., 1999, 14, 722-729). The bisphosphonates may have additional therapeutic uses as it was recently demonstrated that alendronate inhibits the invasion of both prostate and breast cancer cells (Virtanen, S. S., Vaananen, H. K., et al., Cancer Res., 2002, 62, 2708-2714). Finally, a number of nitrogen-containing bisphosphonates have also been shown to inhibit the growth of parasites, including Trypanosoma brucei, Leishmania donovani, and Plasmodium falciparum (Martin, M. B., Grimley, J. S., et al., J. Med. Chem., 2001, 44, 909-916).
There are no GGPP synthase inhibitors currently available for clinical use. There have been several reports of compounds, both synthetic and natural, which inhibit GGPP synthase (Sagami, H., Korenaga, T., et al., Arch. Biochem. Biophys., 1992, 297, 314-320; Szabo, C. M., Matsumura, Y., et al., J. Med. Chem., 2002, 45, 2185-2196; and Zenitani, S., Tashiro, S., et al., J. Antibiot. (Tokyo)., 2003, 56, 617-621). The potency and selectivity of these compounds for GGPP synthase vs. FPP synthase varies significantly. Given the findings discussed above, there is considerable interest in the development of specific GGPP synthase inhibitors.
It would be predicted that selective GGPP synthase inhibitors could be used for the same therapeutic applications as FPP synthase inhibitors as novel anticancer agents, with the added advantage of more specifically affecting the essential downstream targets. That is, while levels of GGPP would be depleted, synthesis of FPP would not be affected, hence the pathways utilizing FPP (e.g., sterol synthesis, dolichol synthesis) would be preserved.
GGPP synthase inhibitors would also serve as important tools which could be used in studies addressing the significance of isoprenoid intermediate pool size, flux through the isoprenoid biosynthetic pathway, hierarchy among geranylgeranylated proteins, and regulatory properties of endogenous isoprenoid pyrophosphates. In specific, while both FPP and GGPP have been shown to regulate the expression of a number of small GTPases (Holstein, S. A., Wohlford-Lenane, C. L. et al., J. Biol. Chem., 2002, 277, 10678-10682; Holstein, S. A., Wohlford-Lenane, C. L. and Hohl, Biochemistry, 2002, 41, 13698-13704; and Holstein, S. A., Wohlford-Lenane, C. L., et al., Biochemistry, 2003, 42, 4384-4391), the relative contribution of the two isoprenoid species has yet to be fully determined. Thus the availability of GGPP synthase inhibitors would provide for novel experimental approaches and improved therapeutic strategies.
In summary there is currently a need for GGPP synthase inhibitors. Such compounds would be useful as chemical tools to ascertain the importance of GGPP in a number of cellular processes. Additionally, they would be anticipated to be useful 1) as antiproliferative agents for the treatment of cancer (based on the FPTase inhibitors), 2) to inhibit testicular function and thus have activity to decrease male fertility (based on high levels in testes), and 3) to treat insulin resistance and obesity based on the ob/ob mouse model of insulin resistance and obesity. They would also be anticipated to be useful in the treatment of a number of parasitic diseases and to have potent osteoclast inhibitory function and to be useful in the prevention and treatment of osteoporosis.